JPS6345372B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antitumor agent for treating tumors with high levels of cytidine deaminase, and more particularly to an antitumor agent that activates 5-trifluoromethyl-2'-deoxycytidine compounds (hereinafter sometimes referred to as F ₃ methyl dC). The present invention relates to the antitumor agent contained as a component. This 5-trifluoromethyl-2'-deoxycytidine functions as a precursor or storage form of the trifluorothymidine compound. 5-Trifluoromethyl-2'-deoxyuridine, or trifluoromidine (F ₃ dT), has already been the subject of research by researchers and has had some success in animal trials. For example, as described in: Y. Fujiwara, T. Oki and C. Heidelberger, “Fluorinated pyrimidines: 5-trifluoromethyl-2′-deoxyuridine for deoxyribonucleic acid synthesis in cultured mammalian cells. ', Mol. Pharmacol. 6 , 273
-280 (1970); C. Heidelberger and SW Anderson, "Tumor suppressive activity of fluorinated pyrimidine XI-5-trifluoromethyl-2'-deoxyuridine", Cancer Research 24 , 1979-1985
(1964): D. Dexter, W. Wahlberg, FJ Ansfield, L. Helson and C. Heidelberger, "Clinical Pharmacology of Trifluoromethyl-2'-deoxyuridine", Cancer Research 32 , 247-
253 (1972); M. Umeda and C. Heidelberger, “Comparative study of fluorinated pyridine and various cell lines”;
Cancer Research 28 , 2529-2538 (1968); C. Heidelberger, J. Boohar and B. Kampshroer, "Fluorinated pyrimidine-5-trifluoromethyluracil-2
``In vivo metabolism of ¹⁴ C and 5-trifluoromethyl-2'-deoxyuridine-2- ¹⁴ C'', Cancer
Researoh 25 , 377-381 (1965); C. Heidelberger, "Mechanism at the molecular level of the antiviral activity of trifluorothymidine", Ann.NYAcad.
Sci. 255 , 317-325 (1975); Y. Fujiwara and C. Heidelberger, "Incorporation of 5-trifluoromethyl-2'-deoxyuridine into deoxyribonucleic acid of fluorinated pyrimidine smallpox virus", Mol.Pharm. 6 , 281-291 (1970); and T. Oki and C. Heidelberger, "Fluorinated pyrimidines - 5-trifluoromethyl-2'-deoxyuridine on the replication of smallpox virus Methsendial ribonucleic acid and proteins."'The action of ',
Mol.Pharm. 7 , 653-662 (1971). However, F ₃ dT is rapidly catabolized, probably because it is a substrate for uridine and thymidine phosphorylases, which remove the sugar moiety of F ₃ dT.
That is, it was found that it degraded into ineffective derivative forms. F ₃ dT shows little selective toxicity to tumors and binds nonspecifically to serum or other proteins. Known as the person responsible for the development of F ₃ dT
Dr. Charles Heidelberger in 1975,
He said he had given up on researching F ₃ dT as a cancer chemotherapeutic agent. Dexter et al. (supra) found that 94% or more of (2- ¹⁴ C)F ₃ dT administered intravenously to patients was excreted in the urine within 48 hours. Ninety percent of the accumulated radioactivity recovered in the urine of patients administered the 6 mg/Kg dose was in the form of trifluorothymine or 5-carboxyuracil. This indicates that F ₃ dT was rapidly degraded. In patients receiving doses below 5 mg/Kg, the F ₃ dT accumulation rate was below 1%.
Catabolic rates were rapid at all doses tested in humans and appeared to be dose-dependent. In 10 to 12 patients in whom an average of 13.5% of the radioactivity was detected in the urine after 1 hour, less than 5% of the radioactivity was due to F ₃ dT and more than 95% was due to trifluorothymine or 5- It was caused by carboxyuracil. The rapid and extensive degradation of F ₃ dT was thought to be caused by nucleoside phosphorylases. Dexter et al.
At least 2 to suppress F ₃ dT catabolism
He suggested that there are two possible methods. The first method consists in adding together with F ₃ dT the drug F ₃ dTMP, which can inhibit degradative enzymes and convert F ₃ dT into its active form. The second method consists in preparing F ₃ dT derivatives that are resistant to phosphorylases and can be converted into nucleosides and nucleotides in vivo. A 90-fold increase in _F3dT dose increased the plasma half-life from 18 to 36 minutes. It was clear to investigators that administration of high doses was undesirable, and low doses were also undesirable because they led to increased catabolism. F ₃ dT was found to be covalently bound to plasma proteins, probably by a mechanism related to interaction with thymidylate synthetase. According to the present invention, a therapeutic agent for leukemia containing an effective amount of 5-trifluoromethyl-2'-deoxycytidine as an active ingredient is provided. Examples of leukemia include acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia. This pharmaceutical preparation is
It is preferred to include a cytidine deaminase inhibitor together with this 5-trifluoromethyl-2'-deoxycytidine. If the tumor to be treated has high cytidine deaminase levels, 5-trifluoromethyl-2'-deoxycytidine is converted to a chemotherapeutic agent (possibly trifluorothymidine) within the tumor over a defined period of time. be done. Therefore, 5-trifluoromethyl-2'-deoxycytidine acts as a cancer-specific storage form of the drug trifluorothymidine, preventing the rapid metabolic degradation of trifluorothymidine found by previous researchers. . Trifluorothymidine is thus formed within tumors where cytidine deaminase levels are high. This trifluorothymidine appears to be formed in relatively small amounts elsewhere in the body;
This is particularly the case when an antineoplastic agent comprising a cytidine deaminase inhibitor, such as tetrahydrouridine, is administered together with -trifluoromethyl-2'-deoxycytidine. The use of cytidine deaminase inhibitors, such as tetrahydrouridine, also reduces _F3 methyl in tumors.
We would like to emphasize that it slows down the dT conversion rate. Thus, the release of trifluorothymidine and its metabolites at the tumor site may vary depending on the cytidine deaminase level of the individual tumor and the amount of tetrahydrouridine or other inhibitor administered. The purpose of cytidine deaminase inhibitors is, of course, protection from serum cytidine deaminase, and the exact dosage is likely to depend on individual serum levels. The F ₃ methyl dC used in the chemotherapeutic agents of the present invention acts as a storage cancer cell inhibitor.
It is believed that this F ₃ methyl dC is converted to 5-trifluorothymidine in the body, which is then metabolized to 5-trifluorodeoxythymidine-5'-phosphate (monophosphate). Until now, cytosine arabinoside (ara-C)
The compounds have been tested as chemotherapeutic agents for cancer.
Some tumors are resistant to ara-C chemotherapy. Such resistance is thought to occur because high levels of cytidine deaminase in the tumor convert ara-C to inactive or less active compounds. On the other hand, since the present invention targets tumors with high cytidine deaminase levels, it can be used to treat tumors resistant to ara-C chemotherapy. As mentioned above, cytidine deaminase in such tumors is thought to convert F ₃ methyl dC to 5-trifluorothymidine by deaminating F ₃ methyl dC at the tumor site. It is well known that cytidine deaminase is present in different concentrations in different parts of the body. The present invention is particularly useful when there are tumors that have significantly higher levels of cytidine deaminase than most other parts of the body. A method for evaluating tissue cytidine deaminase levels was described by Ho and Dah Shi Wang.
Wang), “1-β- in human and mouse tissues”
Distribution of D-arabinofuranosylcytosine kinase and deaminase” [Cancer Res. 33 , 2816-
2820 (1973)]. Ho reports the level of deamination products in nmoles/g tissue/time (for reference, this value is shown below; hereinafter it is also referred to as the "Ho value"). 6000-10000 for peripheral acute lymphoblastic leukemia
It has been reported that peripheral acute myeloid leukemia has a value of 3,500 to 23,000, peripheral chronic myeloid leukemia has a value of 7,300 to 152,000, gastric adenocarcinoma has a value of 9,480, cartilage carcinoma of the leg has a value of 254,500, and Wilms tumor (Wilms' tumor) had a value of 1880.
All of these tumors can be treated, at least in some cases, with the antitumor agents of the present invention. Cytidine deaminase levels were reported to be relatively low in some parts of the body (as shown by the Ho test). Such internal body parts include kidneys (988), brain (44), cerebrospinal fluid (18),
Examples include the heart (972). In contrast, it was reported that normal bone marrow tissue has a value of 16,500 to 86,000, the liver has a value of 6553, and the large intestine mucosa has a value of 1920. For tumors with values above 5000 by the test reported by Ho, cytidine deaminase levels are probably high enough to very strongly suggest that _F3 methyl dC can be used in the treatment of such tumors. . On the other hand, values below 1500 will probably not be considered. Because patients with various types of tumors are expected to have low levels of cytidine deaminase in bone marrow tissue, testing of Ho values in bone marrow tissue is probably standard practice for tumors with values between 1500 and 5000. Dew. In this case, the use of _F3 methyl dC is suggested if the tumor has a higher value than the bone marrow tissue (especially if there is a significant difference). For some types of tumors, _F3 methyl dC
It is expected that the administration of this drug will make the tumor more sensitive to radiation. In such cases, it would be expected to administer 5-trifluoromethyl-2'-deoxycytidine together with 2'-deoxytetrahydrouridine. In most treatments involving the administration of F ₃ methyl dC, it is expected that the F ₃ methyl dC will be administered in conjunction with a cytidine deaminase inhibitor such as tetrahydrouridine or 2'-deoxytetrahydrouridine. If the tumor is the only part of the body with significant cytidine deaminase levels, there is no need to co-administer a cytidine deaminase inhibitor. However, for most uses of the present invention, it is currently expected, however, that tetrahydrouridine will be co-administered before or with the administration of 5-trifluoromethyl-2'-deoxycytidine. If the inhibitor is administered for the first time, it is appropriate to administer it 30 minutes before administration of F ₃ methyl dC. 1/2 of inhibitor 30 minutes before _F3 methyl administration
and the remaining 1/2 of the inhibitor as _F3 methyl
Most preferably, it is administered concurrently with dC administration. The weight ratio of cytidine deaminase inhibitor, such as tetrahydrouridine, to _F3 methyl dC generally ranges from 5:1 to 0.25:1, usually 1:1. 5-trifluoromethyl-2'-deoxycytidine (F ₃ methyl dC), which is the active ingredient of the leukemia therapeutic agent of the present invention, is 5-trifluoromethyl-2'-deoxyuridine (F 3 methyl dC), which has a protected free hydroxyl group. ₃ dU) and ammonia. This manufacturing method is described in JP-A-54-128587. This disclosure is incorporated herein by reference. F ₃ methyl dC can be administered in a variety of ways, including intramuscular, intravenous, topical, and oral, but typically not by intraperitoneal administration. Among these methods of administration, the use of topical application is highly unlikely, although it is generally believed that this technique may be useful for localized treatment of lesions. F ₃ methyl dC is usually administered in combination with a pharmaceutically acceptable carrier or diluent, such as pure saline. Usually _F3 methyl dC is
It is in the form of a pharmaceutical formulation containing F ₃ methyl dC in an amount of 0.01 to about 50% by weight, preferably about 0.05 to 5% by weight. Although relatively high concentrations of F ₃ methyl dC can be applied for topical application, dilute concentrations (5% by weight or less) of F ₃ methyl dC are usually used for intravenous administration. Preferably, F ₃ methyl dC is used at a daily dose of about 250 mg/Kg body weight. When F ₃ Methyl dC is administered once daily, F ₃ Methyl dC is generally administered in an amount of 50 to 250 mg/Kg body weight per day, and treatment is generally continued for several days before the next treatment cycle. There will be a rest period in between. For example, after 5 days of treatment, the patient is allowed to rest for 2 weeks before the next 5-day treatment cycle. If F ₃ methyl dC is administered more than once during the day, the total amount may be up to 500 mg/Kg/day, but it is expected that normally a single daily dose will be used. On the other hand, it is also possible to use a method of administration comprising once weekly administration of F ₃ methyl dC, in which case the dose may be 750 mg/Kg. Single dose 2500mg/Kg _F3 Methyl dC 10000
10% toxicity occurs when used with mg/Kg of tetrahydrouridine. F ₃ methyl dC has F ₃ methyl dC as a main active ingredient and is mixed with a pharmaceutically acceptable carrier or diluent. It can be formulated into pharmaceutical preparations for intraperitoneal administration (for animal studies only), intravenous, subcutaneous, intramuscular, oral or topical administration. The F ₃ methyl dC concentration in such pharmaceutical formulations is
Route of administration, frequency of administration, severity of patient symptoms, age,
It varies in the range of about 0.01 to 50% by weight depending on body weight and general condition. For intravenous injections, the concentration of F ₃ methyl dC is generally about 0.05 to about 5% w/v, and for intramuscular injections it is usually 0.5 to 5% w/v.
It is. The pharmaceutically acceptable carrier or diluent used in this pharmaceutical formulation is the active compound F ₃
Any non-toxic miscible material suitable for mixing with methyl dC may be used. When the pharmaceutical formulation is in a form suitable for intramuscular or intravenous administration, the carrier is preferably an aqueous vehicle, including suspending agents (e.g. methylcellulose or PVP) and/or conventional surfactants. Other conventional additives such as agents may also be included. Despite being members of the same fluorinated pyrimidine family, the administration of F ₃ methyl dC is different from that of 5-fluorouracil (5-FU) and its metabolic precursors. F ₃ methyl dC, unlike 5-FU, is not incorporated into RNA. Also, _F3 methyl
dC differs from 5-FU in 5-FU in RNA.
Does not lead to the formation of fluorocytidine. Furthermore, 5-trifluorodeoxythymidine-5'-phosphate derived from _F3methyldC is 5-trifluorodeoxythymidine-5'-phosphate.
Unlike derivatives of FU, which inhibit thymidylate synthetase, homologs are incorporated into DNA, causing a cessation of DNA synthesis and causing subdivision of m-RNA compared to untreated control group m-RNA. ―
Leads to the formation of RNA. Metabolized 5-FU is
It is incorporated into DNA, but this is a temporary incorporation because there are repair enzymes that remove uracil from the DNA. On the other hand, this does not occur in the case of metabolic derivatives of F ₃ methyl dC. 5-trifluorothymidine triphosphate, derived from F ₃ methyl dC deaminated in tumors,
It is a final product inhibitor of key enzymes involved in synthesis. These enzymes, namely ribonucleoside diphosphate reductase, dCMP deaminase and thymidine kinase, are normally inhibited by thymidine triphosphate, but 5-substituted congeners inhibit these even better than thymidine triphosphate. It was found to inhibit the enzyme. Inhibition of the key enzyme thymidylate synthetase by the 5-FU derivative 5-fluorodeoxyuridylate requires a cofactor, the formation of which is inhibited by methotrexate. For this reason, regimens for the use of methotrexate and 5-FU, often used in combination, are subject to severe limitations. One way to overcome this drawback in 5-FU chemotherapy is to use extremely high concentrations of methotrexate to replace the complexes whose formation is inhibited by methotrexate. However, if F ₃ methyl dC does not require such manipulation in methotrexate co-administration, the conjugate may contain F ₃ methyl dC
The inhibition of thymidylate synthetase by 5-trifluorodeoxythymidine-5'-phosphate derived from By simple 4-amino substitution, F ₃ methyl dC
is very different from trifluorothymidine in terms of metabolic stability, selectivity, and unresponsiveness to titration (ie, incapacitation) by proteins that bind nonspecifically to serum. F ₃ methyl dC is incorporated into DNA by itself when co-administered with 2'-deoxytetrahydrouridine. The incorporation of congeners of both thymidine and deoxycytidine is completely new in chemotherapy. Also, as previously indicated, it has great potential as a motive force for selectively sensitizing tumors to other motivators such as radiation. F ₃ methyl dC alone or F ₃ methyl dC and cytidine deaminase inhibitors were combined with cytoxan and other cytotoxins [e.g.
It may be desirable to use in combination with alkylating agents such as adriamycin and methotrexate or antimitotic agents such as vincristine. Either the tumor fails to react with ara-C or the tumor has high levels of cytidine deaminase by needle biopsy. That is,
The use of F ₃ methyl dC as well as complementary chemotherapy in the treatment of primary tumors may be possible if the Ho value is shown to be at least 1500, especially above 5000. Since F ₃ methyl dC is a storage form of trifluorothymidine, it will also be effective against tumors that respond to F-pyrimidine, regardless of the tumor's cytidine deaminase levels. The results obtained so far suggest that F ₃ methyl dC compounds can also be used for complementary chemotherapy of metastatic or migratory cells. To our knowledge, metastatic cells have not yet been isolated and cytidine deaminase levels in metastatic cells have not been analyzed; however, since cytidine deaminase levels in metastatic cells appear to be relatively high, 5-trifluorocarbon The above method of releasing trifluorothymidine over an extended period of time by administration of methyl-2'-deoxycytidine may be effective. Example 1 Adenocarcinoma 755
Fragments of 2 to 31 mm were implanted subcutaneously in the posterior axilla on both sides of male BDF-1 mice. Six animals were used in each group for the treated and untreated control groups. The animals weighed approximately 22 g at the beginning of the experiment. Treatment groups received F ₃ methyl dC 1, 3, 5, 7 and 9 days after tumor implantation. Each animal in the treatment group received 250 mg/Kg of tetrahydrouridine intraperitoneally, followed 30 minutes later by F ₃ methyl dC250.
mg/Kg was administered intraperitoneally. Drug concentrations were adjusted to reduce body weight on days 7 and 9. Tumor dimensions were measured with calipers and volume was calculated. The results obtained are as follows. Tumor volume 10th day Treatment group/control group <. 01 Day 12 Treatment group/control group =. 015 Day 16 Treatment group/control group =. 088 Day 19 Treatment group/control group =. 28 Weight loss Day 7 Treatment group -4.18g Control group -1.24g Day 9 Treatment group -5.1g Control group +2.0g Day 12 Treatment group -5.2g Control group +3.4g Around day 15, The treated group of animals no longer showed signs of severe toxicity. After 25 days, the poisoning death was not observed at all. Example 2 Adenocarcinoma 755 The procedure of Example 1 was repeated. However, _F3 methyl dC and tetrahydrouridine are effective against tumor metastasis.
Administration was performed 3.5, 7, 9 and 11 days later. The weight of the animals used in this experiment was 24 g at the beginning of the experiment. Five animals were used in each group. The drug concentration is 7,
Adjustments were made to reduce body weight on days 9 and 11. The results obtained in this experiment are as follows. Tumor volume Day 11 Treatment group/control group =. 19 Day 13 Treatment group/control group =. 17 Day 15 Treatment group/control group =. 23 Weight loss 7th day Treatment group -4.18g Control group +1.24g 9th day Treatment group -5.2g Control group +1.69g 11th day Treatment group -5.36g Control group +2.18g No poisoning deaths after 25 days It was not observed. _F3
Animals treated with methyl dC showed signs of reduced toxicity after 14 days. Example 3 Sarcoma 180 Ascites Tumor In this experiment, only a single injection of F ₃ methyl dC was used [see J. Bertino et al.
"Anticancer effects dependent on dosage regimen", Cancer Res. 37 ,
327-328 (1977)]. 10 ⁵ Sarcoma 180 ascites tumor cells were injected intraperitoneally into female Swiss mice, followed by a single dose of the indicated compound 3 days after tumor inoculation. Methotrexate is 5
- Administered 2 hours before fluorouracil or F ₃ methyl dC administration, and tetrahydrouridine (H ₄ U) 30 minutes before F ₃ methyl dC administration.
The results obtained are as follows.

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Claims (1)

[Scope of Claims] 1. A therapeutic agent for leukemia containing an effective amount of 5-trifluoromethyl-2'-deoxycytidine as an active ingredient. 2. The therapeutic agent for leukemia according to claim 1, wherein trifluorothymidine is formed in a tumor by conversion of 5-trifluoromethyl-2'-deoxycytidine. 3. The therapeutic agent for leukemia according to claim 2, wherein the conversion is deamination by cytidine deaminase. 4. The therapeutic agent for leukemia according to claim 3, wherein the tumor is sensitive to trifluorothymidine. 5. The therapeutic agent for leukemia according to claim 4, wherein the tumor has ara-C resistance. 6. The therapeutic agent for leukemia according to claim 3, wherein the tumor is sensitive to a fluorinated pyrimidine compound. 7. The therapeutic agent for leukemia according to claim 6, further comprising an inhibitory amount of a cytidine deaminase inhibitor in addition to 5-trifluoromethyl-2'-deoxycytidine. 8. The therapeutic agent for leukemia according to claim 7, wherein the cytidine deaminase inhibitor is tetrahydrouridine or 2'-deoxytetrahydrouridine. 9. The therapeutic agent for leukemia according to claim 8, wherein the weight ratio of the cytidine deaminase inhibitor to the 5-trifluoromethyl-2'-deoxycytidine is about 5:1 to about 0.25:1. 10. The therapeutic agent for leukemia according to claim 9, wherein at least a portion of the cytidine deaminase inhibitor is taken before taking 5-trifluoromethyl-2'-deoxycytidine. 11. The therapeutic agent for leukemia according to claim 7, wherein the daily dose of 5-trifluoromethyl-2'-deoxycytidine is about 50 to about 500 mg/Kg body weight. 12. The therapeutic agent for leukemia according to claim 11, wherein the daily dose is about 250 mg/Kg body weight. 13 Tumor is at least 1500 H. The therapeutic agent for leukemia according to claim 1, which has the following value. 14H. The therapeutic agent for leukemia according to claim 13, which has a value of 5000 or more.